The aim of this study was a structural characterization of the binding of tetrahydromethanopterin derivatives to enzymes of the energy conserving CO2-reducing methanogenic pathway. In this pathway the stepwise reduction

The aim of this study was a structural characterization of the binding of tetrahydromethanopterin derivatives to enzymes of the energy conserving CO2-reducing methanogenic pathway. In this pathway the stepwise reduction of CO2 proceeds via binding to the tetrahydrofolate analog tetrahydromethanopterin (H4MPT) which is found i. a. in methanogenic archaea. Due to the adaptation of the thermophilic and hyperthermophilic source organisms (Methanothermobacter marburgensis, Methanocaldococcus jannaschii and Methanopyrus kandleri) to their extreme habitats by genomic, structural and enzymatic features, they are of special interest for structural biology. The first enzyme investigated in this study is the eight subunits containing membrane bound complex of N5-methyl-H4MPT:coenzyme M methyltransferase (MtrA-H). Firstly, it catalyzes the methyl group transfer from H4MPT to the Co(I) of the prosthetic group (5’-hydroxybenzimidazolylcobamide; vitamin B12a). In a second step, it transfers the methyl group to coenzyme M, which is coupled to an energy conserving vectorial sodium ion transport across the membrane. The purification protocol for Mtr complex from M. marburgensis (670 kDa) previously established under anaerobic conditions was enhanced, simplified for isolation and purification under aerobic conditions and optimized for electron microscopic single particle reconstruction. Besides the preparation of the complete complex MtrA-H, the preparation of enzyme complex MtrA-G without the most hydrophilic subunit MtrH was chosen as a second approach. The purification method developed for this purpose improved the control over dissociation of MtrH from complex MtrA-G and enhanced the homogeneity of the sample significantly. Thus, the prerequisites for crystallization and subsequent X-ray studies were created as well as for electron microscopic single particle reconstruction, which was confirmed by experiments with MtrA-G (without MtrH) promising far better results. Concurrently to the studies on the complete Mtr complex, cobamide containing subunit MtrA and H4MPT binding subunit MtrH should be purified to homogeneity in quantities sufficient for crystallization and X-ray analysis. Therefore, MtrA and MtrH from source organisms mentioned above were cloned for heterologous expression in E. coli, expression conditions were optimized and purification protocols were established. The purified proteins were used for extensive crystallization experiments. MtrA from M. jannaschii without its transmembrane helix could be produced in E. coli as a soluble protein. The holoprotein could be purified to homogeneity but crystallization failed presumably due to its exceptionally high solubility. MtrA from M. kandleri was produced in E. coli as a StrepII fusion protein without transmembrane helix only in marginal amounts. The production of subunit MtrH in E. coli as a soluble protein was not possible regardless of the variants tested in this thesis. Attempts to refold and purify to homogeneity the M. marburgensis protein expressed in inclusion bodies were without success. Co-expression of MtrA and MtrH with the objective of improving folding and solubility also led to the production of inclusion bodies which could not be refolded and purified together. The second enzyme analyzed in this thesis, F420-dependent N5,N10-methylene-H4MPT dehydrogenase (Mtd), catalyzes the reversible stereospecific hydride transfer between reduced F420 (F420H2) and methenyl-H4MPT+, the latter being thereby reduced to methylene-H4MPT. The ternary complex involved in this reaction consists of the protein part, substrate (methylene-H4MPT) and co-substrate (F420) and was structurally characterized in this thesis. The purified recombinant enzyme from M. kandleri was co-crystallized with several H4MPT and F420 derivatives, the structure of the ternary complex was determined by X-ray crystallography and the binding of H4MPT and F420 was analyzed. In the structure solved in the thesis methenyl-H4MPT+ and F420H2 are bound in a catalytically active conformation, but a resolution of 1.8 Å precludes a discrimination between either methylene-H4MPT and F420 or methenyl-H4MPT+ and F420H2. Compared to the structure of M. kandleri Mtd (KMtd) without substrate and co-substrate bound, only marginal variations of the protein conformation were visible. Thus KMtd can be considered as a surprising and extreme example of an enzyme with an exceptionally rigid, preformed binding pocket.